For many years now collagenous tissues have been a source of materials implanted as bioprosthesis, used on the surface of the body as tissue dressings, or as carriers in many biological applications. The source of such materials and the materials in question include porcine heart valves, bovine ligaments and tendons, bovine and human blood vessels, pericardial patches, demineralized bone matrix, suture materials made out of intestinal serosa or reconstituted collagen, among others. Collagen prepared from such tissues using various methods and exposed to various degrees of purification are used as hemostatic agents, tissue fillers and expanders, drug carriers, dermal substitutes, ophthalmic dressings, etc. The collagen in these preparations varies in its grade of purity, as well as in the degree in which it resembles the native configuration (molecular packing fiber orientation, fiber diameter) and retention of native configuration (triple-helix) found in the live tissue.
The native characteristics of the collagen network are summarized schematically in FIG. 1. The individual molecules, which appear as very long rods, are composed of 3 chains known as .alpha. chains. For such molecules to retain their fundamental biological properties it is essential that the 3 .alpha. chains remain in a triple helical conformation. The non helical regions which are attached to the ends of the molecules are not essential for this purpose, but can be rather detrimental for the application cited as they contribute very significantly to generate immunological problems. These non-helical regions, remain on the surface as well as buried within the fibers when such molecules assemble to form such fibers. Prior technology was unable to remove them without disrupting the fibrillar structure. In FIG. 1 these terminal extensions (telopeptides) are shown as appendages at the ends of the molecules.
The method described allows for enzymes to reach such arenas of the fibril and remove these non-helical extensions without dissociating the fibers into individual molecules and causing the fibrils to disassemble.
In essence, antigenic determinants can be removed, as well as other contaminating extraneous molecules, which exist between the collagen while leaving the fibrils essentially intact. Therefore such fibers will retain their original characteristics (i.e. diameter, length, orientation within the matrix, packing density, surface characteristics, etc.) such organization, identified as a native structure of configuration, is essential for the collagen network to exhibit its desired properties (i.e. mechanical function, structural support, cell surface compatibility, etc.).
In addition, it is essential for the individual molecules to also retain their native configuration (triple helix). Prior technology did not allow all of these features to be retained simultaneously, either one or another (native packing or molecular integrity) had to be lost. This invention allows for the essential characteristics of collagen to be retained.
In the prior art, when tissues such as porcine heart valves are used as bioprosthesis, the relevant area of the animal is explanted, crosslinked in its native configuration with agents such as glutaraldehyde or other crosslinking reagents, and after mounting on suitable stents, implanted into the host. Such implants contain not only the structural framework of collagen but large numbers of substances, not necessary for structural purposes but which are associated with the donor tissue, i.e. cells and cell debris, interfibrillar matrix, elastin, etc. Some of these substances are not as readily tanned as collagen and may preferentially leach out following implantation into the host, giving rise to a series of undesired reactions (i.e. immunological sensitization, foreign body tissue reaction, etc.).
In the prior art attempts were made to remove these contaminants without effecting the collagen framework but these have proven unsuccessful. Either the collagen becomes degraded or the telopeptide free molecules become dispersed in the solution. If the collagen is crosslinked prior to chemical or enzymatic removal of these noncollagenous substances, the collagen becomes irreversibly and to such impurities. The crosslinking reagents which do not selectively react with collagen, but also interact with many of these less desirable molecules, render them also insoluble and impossible to remove without disrupting the collagenous framework. The need to eliminate these noncollagenous substances while leaving behind the collagen fibrils in their native 3-dimensional configuration within the tissue is therefore well recognized. The method we describe provides the necessary scaffolding for such implants to function biomechanically in a tissue and organ compatible manner, but devoid of non-essential components.
In a corollary of the procedure developed, we have learned how to generate a purified collagenous network which can also serve as a source of purified collagen for a variety of uses.
Based on the method described the collagen can be formulated as a hemostatic agent with several intrinsic advantages over other collagenous materials devoted to similar use. It retains the native fibrillar characteristics, which are known to be essential for platelet aggregation to occur, the native fibrillar diameter and is devoid of other impurities that can hinder the recognition of the collagen surface by platelets, cells or other molecular species. A hemostatic agent as described here is a preparation of collagen which is able to aggregate platelets on its surface and initiate the process of clot formation.
A material can also be prepared which has ideal suture characteristics, and which is compatible with the process of wound healing. A surgical suture is a collagen derived material, which has thread like characteristics, which is derived from animal tissues such as intestinal serosa and which is used to close open wounds or repair organ defects. The material in question retains the desired mechanical properties, since the collagen framework is left intact by the treatment received, while impurities associated with the natural tissues have been removed. This makes the collagenous material much more biocompatible, and less likely to elicit the immunological response associated with the large number of immune related cells which populate wounded areas. The enzymatic treatment also renders the collagen more visible to tissue proteases and therefore allows for its more rapid removal. The removal of impurities which can mask reactive groups on collagen can allow the process of resorbtion of such sutures to be more adquately modulated by the process of crosslinking. The introduction of chemical crosslinks can therefore be performed in a much more controlable fashion.
In essence, the process described generates a purified network of collagen which can be crosslinked using standard bifunctional agents or other forms of crosslinking methods, or can be used as a starting material for the purification of collagen in large amounts. In the latter case, the final product can present itself in the form of native fibrils or monomeric collagen after suitable dispersion of the collagenous network. The fact that one can retain the native collagenous framework, in its 3-dimensional array devoid of surrounding non-collagenous materials is the essence of our discovery. This cannot be achieved by the prior art.